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  • The Forkhead Transcription Factor FOXM1 Controls Cell Cycle- Dependent Gene Expression through an Atypical Chromatin Binding Mechanism

    Xi Chen,a Gerd A. Müller,b Marianne Quaas,b Martin Fischer,b Namshik Han,a Benjamin Stutchbury,a Andrew D. Sharrocks,a

    Kurt Engelandb

    Faculty of Life Sciences, University of Manchester, Manchester, United Kingdoma; Molecular Oncology, Medical School, University of Leipzig, Leipzig, Germanyb

    There are nearly 50 forkhead (FOX) transcription factors encoded in the human genome and, due to sharing a common DNA binding domain, they are all thought to bind to similar DNA sequences. It is therefore unclear how these transcription factors are targeted to specific chromatin regions to elicit specific biological effects. Here, we used chromatin immunoprecipitation fol- lowed by sequencing (ChIP-seq) to investigate the genome-wide chromatin binding mechanisms used by the forkhead transcrip- tion factor FOXM1. In keeping with its previous association with cell cycle control, we demonstrate that FOXM1 binds and regu- lates a group of genes which are mainly involved in controlling late cell cycle events in the G2 and M phases. However, rather than being recruited through canonical RYAAAYA forkhead binding motifs, FOXM1 binding is directed via CHR (cell cycle genes homology region) elements. FOXM1 binds these elements through protein-protein interactions with the MMB transcrip- tional activator complex. Thus, we have uncovered a novel and unexpected mode of chromatin binding of a FOX transcription factor that allows it to specifically control cell cycle-dependent gene expression.

    There are nearly 50 different forkhead transcription factors en-coded in mammalian genomes, and these proteins all contain the conserved forkhead DNA binding domain (reviewed in refer- ences 1 and 2). Forkhead transcription factors are involved in controlling a wide range of biological processes and are aberrantly expressed or regulated in disease states, including cancer (re- viewed in reference 2). However, due to sharing a common DNA binding domain, forkhead transcription factors are generally be- lieved to bind to variations of the RYAAAYA motif. Hence, it is unclear how individual forkhead proteins are specifically re- cruited to the regulatory regions of different cohorts of target genes to control defined biological responses. One key process which is controlled by forkhead transcription factors is the cell cycle and, in particular, the G2-M transition. The initial links to G2-M control were made with the Saccharomyces cerevisiae fork- head protein Fkh2, which controls the temporal expression of a cluster of genes at this phase of the cell cycle (reviewed in reference 3). More recently, members of the FOXO and FOXM classes of forkhead transcription factors have been linked with controlling the same process in mammalian cells (4–6). In both cases, fork- head transcription factors coordinate the integration of signals from the cell cycle regulatory machinery to transcriptional out- puts. This is exemplified by the links to the cell cycle regulated Polo-like kinase PLK1, which is recruited to cell cycle-regulated promoters through promoter elements bound by the forkhead transcription factors FOXM1 and Fkh2, albeit indirectly in the case of Fkh2 (7, 8).

    In mammalian cells, the transcriptional control of a cluster of genes at the G2-M transition, is coordinated through promoter elements which typically contain CHR (cell cycle genes homology region) and CDE (cell cycle-dependent element) motifs. In addi- tion, there are usually closely associated CCAAT boxes for the recruitment of the NF-Y transcription factor (reviewed in refer- ence 9). The CHR is typically located at or close to the transcrip- tional start site. Recently, it was shown that the CHR element is

    bound by the DREAM and MMB transcriptional regulatory com- plexes, and these complexes play a role in controlling cell cycle- dependent transcription of genes expressed at the G2-M border (10). The DREAM and MMB complexes are functionally inter- related, and both contain the MuvB core complex, which includes LIN9, LIN37, LIN52, LIN54, and RBBP4 in addition to specific additional subunits in each case (11, 12). The DREAM complex is repressive in nature and contains additional subunits such as p130 and E2F4, whereas the MMB complex is thought to activate tran- scription and contains B-MYB (MYBL2). Although FOXM1 is known to play a role in controlling the expression of the same class of genes as bound by the DREAM and MMB complexes, it is unclear how these complexes interact. Indeed, there appear to be no conserved canonical forkhead binding motifs within proximal promoters of genes expressed during G2/M, that correspond to the classic RYAAAYA motif recognized by most members of this tran- scription factor family. Several studies have implicated upstream forkhead binding motifs in mediating the response of a subset of G2-M genes to FOXM1 (see, for example, reference 6), but this has not been systematically investigated across all of the genes acti- vated during this part of the cell cycle.

    Here, we have used chromatin immunoprecipitation followed

    Received 29 June 2012 Returned for modification 21 August 2012 Accepted 24 October 2012

    Published ahead of print 29 October 2012

    Address correspondence to Andrew D. Sharrocks, [email protected]

    Supplemental material for this article may be found at /MCB.00881-12.

    Copyright © 2013, American Society for Microbiology. All Rights Reserved.


    The authors have paid a fee to allow immediate free access to this article.

    January 2013 Volume 33 Number 2 Molecular and Cellular Biology p. 227–236 227

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  • by sequencing (ChIP-seq) to interrogate the chromatin binding profile of FOXM1 in a genome-wide manner. In keeping with its known role in cell cycle control, FOXM1 is generally bound to the regulatory regions of genes which encode proteins involved in mitotic regulation. However, surprisingly, FOXM1 binding is not associated with forkhead DNA binding motifs but instead coin- cides with CHR elements. Rather than binding to the CHR di- rectly, FOXM1 interacts with the MMB complex and is recruited to chromatin via interactions with this complex. Thus, FOXM1 has an atypical mode of chromatin association which distin- guishes it from other family members and allows it to have a spe- cific role in cell cycle control.

    MATERIALS AND METHODS Plasmid constructs. The human CCNB1 promoter reporter plasmids pAS3017 (pGL4.10-hCCNB1 WT) and pAS3019 (pGL4.10-hCCNB1 CHR mutant) were derived from hB1-Luci constructs that have been described earlier (13). pRL-CMV-vector (Promega), pAS188 (pCMV5 empty vector), and pAS1175 (pCMV5-FOXM1b) are used in luciferase assays. pAS1175 (encoding FOXM1b with Flag and hexahistidine C-ter- minal tags) was constructed by a two-step procedure, first by cloning a HindIII/XhoI-cut PCR product (primers ADS1177/ADS1178 on tem- plate IMAGE clone 3834244) into pAS728 to create pAS1171, followed by cloning a HindIII/XbaI fragment from pAS1171 into the same sites in pCMV5. The plasmids pAS3048 (p3�Flag-FOXM1b-WT) (a gift from Suyun Huang) and pAS3069 [p3�Flag-FOXM1b(�1-116)] were used in ChIP assays. The plasmids used in glutathione S-transferase (GST) pulldown experiments were pAS3059 [GST-FOXM1b(1-367)], pAS3060 [GST-FOXM1b(117-367)], pAS3061 [GST-FOXM1b(235-367], pAS3062 [GST-FOXM1b(235-490)], pAS3068 [GST-FOXM1b(451-748)], pAS3066 [GST-FOXM1b(1-130)], and pAS3067 [GST-FOXM1b(1-235)], which were generated by ligating EcoRI/XhoI-cut PCR products generated with the oligonucleotide pairs ADS2908/2911, ADS2909/2911, ADS2910/2911, ADS2910/2912, ADS2932/2913, ADS2908/2918, and ADS2908/2919, re- spectively, into pGEX-6p1 vector (GE Healthcare).

    Tissue culture, transfection, reporter assay, and reverse transcrip- tion-PCR. U2OS, HEK293/293T, NIH 3T3, and HCT116 cells were main- tained in Dulbecco modified Eagle medium containing 10% fetal bovine serum. To block cells at G1/S, U2OS cells were cultured in medium con- taining 2 mM thymidine (Sigma; catalog no. T1895) for 16 h, followed by a release in fresh medium for 12 h and then in medium containing 2 mM thymidine for another 16 h.

    Lipofectamine RNAiMAX (Invitrogen) transfection reagent was used for small interfering RNA (siRNA) transfection; X-tremeGene HP DNA (Roche) and Polyfect (Qiagen) transfection reagent were used for plasmid transfections. At 24 to 48 h after transfection, the cells were harvested for further analyses. All siRNAs were ON-TARGETplus SMART pools from Dharmacon, and a final concentration of 20 nM was used, except for the siRNA against mouse FoxM1, which was described previously (6). Con- trol nontargeting siRNAs (Dharmacon) were used throughout. For the luciferase assays, 12-well plates were used, and for each well the cells were transfected with 200 ng of reporters containing the appropriate promot- ers, 10 ng of Renilla luciferase plasmid pGL4.70, and 790 ng of empty or FOXM1 expression plasmids. At 24 h after transfection, NIH 3T3 cells were synchronized in G2/M by serum deprivation for 60 h, followed by restimulation as described previously (10), and harvested for a luciferase assay using a dual-luciferase reporter assay system (Promega) according to the manufacturer’s instructions. Real-time reverse transcription-quan- titative PCR (RT-qPCR) was carried out as described previously (14). The primer-pairs used for RT-qPCR exp

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